Fig 2: Inactivation of STUB1 or PTPN2 sensitized human tumour cells to growth inhibition induced by cytokines. (a) Western blot analysis of the expression level of PTPN2 and STUB1 in tumour cells. (b) Flow cytometry plot showing the surface expression level of IFNGR1 in tumour cells. In parallel, parental cells were stained with PE-conjugated isotype control antibody to demonstrate low level of non-specific binding. (c) Level of phosphorylated Tyr701-STAT1 after 30-min response to varying doses of IFN? as measured by Lumit immunoassay. (d) Fold change (FC) in ATP level relative to the parental cells as a quantification of viable cells. Measurements were performed using CellTiter-Glo 2.0 assay after 6-day treatment with the cytokines (10 ng ml-1 each). Data are mean ± s.d. from two biological replicates (c) or mean ± s.e.m. from three biological replicates (d). P values were determined by ordinary two-way ANOVA on Log2-transformed data with Dunnett’s multiple comparisons test versus parental cells, **P = 0.01, ****P = 0.0001, ns P > 0.90 (d). Representative of two independent experiments (a–d).

Fig 3: Stub1 deletion enhances antigen processing and presentation by sensitizing tumour cells to IFN?. (a–c) Flow cytometry analysis of cell surface MHC-I on parental, control or independent Stub1-null B16-F10 cells. gMFI, geometric mean fluorescence intensity. (d, e) Western blot analysis of the expression level of STUB1, STAT1, STAT2, IRF1, PSMB8, PSMB9 and PSMB10 in parental, control or independent Stub1-null B16-F10 cells. Band intensity was normalized with total protein signal. The tumour cells were either untreated (Nil) or treated with IFN? for 24 h (a–e). See Supplementary Fig. 1 for additional flow cytometry plots, Western blot data and analysis (a, d, e). (f) Western blot analysis of the expression level of IRF1, STAT1, and phosphorylation of Tyr701-STAT1 at 2 h post-treatment with IFN? (twofold serial dilution from 2.0 ng ml-1). (g) Volcano plot showing differential presentation of MHC-associated peptide in gStub1 #1 versus gControl cells, following stimulation with 0.03 ng ml-1 IFN? for 24 h. Red circles highlight peptides significantly enriched in gStub1 #1 cells (twofold cutoff, P = 0.01; n = 3 biological replicates). FC, fold change. See Supplementary Fig. 2d for data of gStub1 #2 cells. Representative of four (a) or two (d–f) independent experiments. Data are mean ± s.d. (b) or mean with all data points (c) from four independent experiments. P values were determined by ordinary two-way ANOVA on Log2-transformed data with Dunnett’s multiple comparisons test, ****P = 0.0001 (c).

Fig 4: Stub1 and Chic2 KO lead to increased endogenous total and cell surface Csf2rb.A, Western blots of 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 for STUB1 and Vinculin. B, Western blots of 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 for Chic2 and Vinculin. C, Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 cultured in 5, 0.1, or 0.01 ng/ml IL-3 (S.E. = short exposure, L.E. = long exposure). Representative of three independent biological replicates with similar results. D, Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 cultured in 5, 0.1, or 0.01 ng/ml IL-3. Representative of three independent biological replicates with similar results. E, Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 treated with 10 µM cycloheximide (CHX) for 0, 2, 4, or 8 h. F, Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 treated with 10 µM cycloheximide (CHX) for 0, 2, 4, or 8 h. G, Western blots of Csf2rb, Stub1, Chic2, and Vinculin in 32D Cas9 cells with sgNT/sgNT, sgStub1-1/sgNT, sgNT/sgChic2-1, or sgStub1-1/sgChic2-2. H, bar graph showing ratio of mean GFP to mean mCherry signal of Csf2rb reporter in 32D cells with sgNT, sgChic2-1/2, or sgStub1-1/2 in 0.01 ng/ml IL-3 as measured by flow cytometry. Bars show GFP/mCherry ± SD from n = 3 replicates. I, bar graph showing median fluorescence intensity for anti-Csf2rb-PE (CD131) in 32D Cas9 cells with sgNT, sgChic2-1/2, or sgStub1-1/2 in 0.01 ng/m IL-3 as measured by flow cytometry. Bars show mean ± SD from n = 3 replicates. p-values calculated by unpaired Student’s t test between sgNT and other conditions at each cytokine concentration.

Fig 5: Pharmacological inhibition of STUB1 with expressed biologic phenocopies the genetic knockout. (a, b) Validation of the binding of synthetic peptides to STUB1 (aa25–aa153) by isothermal titration calorimetry (a) and thermal shift assay (b). Representative of three independent experiments (a). Data are mean ± s.d. of six replicates derived from three independent experiments (b). (c) Competitive fluorescence polarization assay. Synthetic peptides were assessed for their ability to compete with 15 nM of tracer peptide (5-FAM-SSGPTIEEVD) for binding to 1 µM STUB1 (aa25–aa153). Data are mean ± s.d. of six replicates derived from two independent experiments. (d) Design of the inhibitory biologic by grafting the peptide (SIWWPD) to the C-terminus of an mCherry2 (red) scaffold. The fused peptide blocks the function of the tetratricopeptide repeat domain (blue) of STUB1 (PDB code 2C2L) and inhibits its substrate binding. U-box domain (orange) which recruits the E2 ubiquitin-conjugating enzyme is not affected. (e) Generation of tumour cell lines stably expressing the biologic or its control. Plasmid encoding the biologic was electroporated into tumour cells, followed by antibiotics selection of the stable clones. The mCherry2-positive cells (red dotted box) were further gated for mCherry2hi population (top 50th percentile, red box). Gating example represents IFN?-treated B16-F10 stable cell lines. (f, g) Flow cytometry analysis of the relative cell surface level of IFNGR1 (f) and MHC-I (g) expressed by the mCherry2hi population in B16-F10, A375 or A549 cells. The cells were either untreated or treated with mouse IFN? (0.03 ng ml-1) or human IFN? (0.01 ng ml-1) for 24 h. The expression levels were normalized to the average value of the control (mCherry2-SIWWHR). n = 5 biological replicates from two independent experiments (f–g). Bars are mean with all data points (f–g). P values were determined by ordinary two-way ANOVA in each cell type with Sidak’s multiple comparisons test, ****P = 0.0001 (f–g). (h) Co-immunoprecipitation (co-IP) of FLAG-mCherry2-peptide and STUB1 from the cellular lysate of B16-F10 using anti-FLAG antibody. Synthetic peptide (SIWWPD) was added into the co-IP mixture to assess specificity of the interaction. Blot is representative of three independent experiments.